Ginsenoside derivatives, preparation methods, and anti-inflammatory applications thereof

Abstract

The present invention discloses ginsenoside derivatives represented by Formula I, II, III, IV, V, or VI, their pharmaceutically acceptable salts, and methods for preparing the same. The ginsenoside derivatives represented by Formula I, II, III, IV, V, or VI and their pharmaceutically acceptable salts provided herein possess anti-inflammatory activity and can be used to prepare anti-inflammatory drugs or anti-inflammatory pharmaceutical compositions. Compared to existing clinical drugs, the ginsenoside derivatives provided herein exhibit superior efficacy in inhibiting LPS-induced NO release, and exhibit excellent safety, reduced toxicity, and enhanced stability. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the fields of organic synthesis and medicinal chemistry, and in particular to a class of ginsenoside derivatives, a preparation method thereof, and an application thereof in the preparation of anti-inflammatory drugs, belonging to the technical field of proposal, preparation and application of new compounds. Background Art

[0002] Inflammation is the body's defensive response to stimuli, manifesting as redness, swelling, heat, pain, and dysfunction. Normally, inflammation is beneficial, acting as the body's automatic defense response. However, excessive stimulation from inducing factors can lead to an immune overreaction, resulting in tissue damage and even life-threatening consequences.

[0003] Currently, clinically used anti-inflammatory drugs all have adverse reactions to varying degrees. Therefore, the development of highly effective and low-toxic anti-inflammatory drugs is a current research focus.

[0004] Nitric oxide (NO) is a small molecule with specialized biological functions. Gaseous NO can freely transport biological membranes, transmitting biological information and acting as a crucial intracellular messenger. It is primarily produced by inducible nitric oxide synthase (iNOS), which is not expressed in normal tissues but is overexpressed in inflamed tissues. Its level serves as an important indicator of inflammation severity and plays a crucial role in regulating inflammation, immunity, and cancer.

[0005] During inflammation, mitochondrial-derived ROS (MtROS) contribute to the production of various proinflammatory cytokines. The MitoSOX Red Mitochondrial Superoxide Indicator (MitoSOX Red Mitochondrial Superoxide Indicator), a cationic derivative of dihydroethidum (DHE), can highly specifically detect the production of MtROS in living cells. In macrophages, another important function of MtROS is regulating the inflammasome. The NLRP3 inflammasome is known to sense various inflammatory stimuli through the release of IL-1β and IL-18, promoting the development of inflammation and thereby causing further damage, such as the development of neurodegenerative diseases and type 2 diabetes. MtROS is an important stimulus for activating the NLRP3 inflammasome.

[0006] Ginseng ranks first among traditional Chinese medicines, boasting multiple medicinal benefits, including calming the mind and prolonging life. Ginsenosides, the primary active components of ginseng, possess a wide range of pharmacological actions. Ginsenosides Rb1, Re, Rg1, Rg3, and Rh1, among others, have demonstrated significant anti-inflammatory activity, exhibiting significant therapeutic effects against neuroinflammation, skin inflammation, and colitis. Pyxinol and its C-24 diastereomer are key metabolites of 20S-protopanaxadiol in the body and are more readily absorbed by the body than the parent ginsenosides. Previous studies have found that Pyxinol can be prepared in large quantities through the direct cyclooxidation of 20(S)-protopanaxadiol (Chin.J.Org.Chem.37(2017), 2109-2114), and that its esterified derivatives have inhibitory effects on proinflammatory factors NO and TNF-α (patent application publication numbers CN 109776647A and CN 111647036A). However, due to the presence of ester groups, the esterified derivatives disclosed in the prior art are easily hydrolyzed by esterases in plasma, resulting in poor drug stability in plasma. In order to obtain anti-inflammatory compounds with better activity and greater stability in the body, the present invention has carried out a new structural transformation and modification to screen and obtain anti-inflammatory drug candidates with greater development value. Summary of the Invention

[0007] The purpose of the present invention is to provide a ginsenoside derivative and its preparation method and anti-inflammatory application.

[0008] In order to solve the above problems, the present invention provides the following technical solutions:

[0009] Ginsenoside derivatives as shown in formula I, II, III, IV, V or VI, or pharmaceutically acceptable salts of ginsenoside derivatives as shown in formula I, II, III, IV, V or VI

[0010]

[0011] In formula I, II, III, IV, V or VI, R is 4-nitrophenyl, 4-nitrilephenyl, 4-trifluoromethylphenyl, 4-bromophenyl, 4-methylphenyl, 3-pyridyl, phenyl or cyclohexyl, preferably 4-nitrophenyl, 4-nitrilephenyl or 3-pyridyl.

[0012] Preferably, the ginsenoside derivative is 20(S)-12β, 20-dihydroxy-2-(4-nitrobenzylidene)-dammarane-24-ene-3-one, 20(S)-12β, 20-dihydroxy-2-(4-cyanobenzylidene)-dammarane-24-ene-3-one, 20(S)-12β, 20-dihydroxy-2-(3-pyridylmethylene)-dammarane-24-ene-3-one, (20S, 24R)-epoxy-12β, 25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one, (20S, 24R)-epoxy-12β, 25-dihydroxy-2-(4-cyanobenzylidene)-dammarane-3-one, (20S, 24R)-epoxy- 12β,25-dihydroxy-2-(3-pyridylmethylene)-dammarane-3-one, (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one, (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one, 20(S)-dammarane-2-(4-nitrobenzylidene)-24-ene-3β-12β,20-triol, 20(S)-dammarane-2-(4-nitrobenzylidene)-24-ene-3β-12β,20-triol or (20S,24R)-epoxydammarane-2-(4-nitrobenzylidene)-3β,12β,25-triol.

[0013] The present invention also provides a method for preparing the ginsenoside derivatives shown in Formula I, II, III, IV, V or VI. The synthetic route of the method is shown in the following formula:

[0014]

[0015] Furthermore, the method comprises the following steps:

[0016] (1) Protopanaxadiol (20S-PPD), the 24R-pyxinol ginsenoside derivative shown in Formula 3, or the 24S-pyxinol ginsenoside derivative shown in Formula 4 are subjected to a selective oxidation reaction under the action of an oxidant to prepare 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one shown in Formula 1, the intermediate product shown in Formula 5, or the intermediate product shown in Formula 7, respectively;

[0017] The oxidizing agent is Dess-Martin reagent (DMP reagent) or PCC (pyridinium chlorochromate), preferably DMP reagent.

[0018] The 24R-pyxinol ginsenoside derivative shown in formula 3 is (20S, 24R)-epoxydammarane-3β, 12β, 25-triol, and the 24S-pyxinol ginsenoside derivative shown in formula 4 is (20S, 24S)-epoxydammarane-3β, 12β, 25-triol.

[0019] Further, the solvent of step (1) is dichloromethane (DCM);

[0020] The volumetric amount of dichloromethane is generally 10 to 40 mL / g, preferably 20 to 25 mL / g, based on the mass of protopanaxadiol (20S-PPD), the 24R-pyxinol ginsenoside derivative shown in Formula 3, or the 24S-pyxinol ginsenoside derivative shown in Formula 4.

[0021] The molar ratio of protopanaxadiol (20S-PPD), the 24R-pyxinol ginsenoside derivative shown in Formula 3 or the 24S-pyxinol ginsenoside derivative shown in Formula 4 to DMP is preferably 1:1 to 1.2;

[0022] The reaction temperature is room temperature, and the reaction time is 3 to 8 hours.

[0023] When the oxidant is DMP reagent, sodium bicarbonate is added to adjust the pH, and the mass ratio of DMP reagent to sodium bicarbonate is 1:0.3-1.

[0024] When the oxidant is DMP, an appropriate amount of tert-butyl alcohol is added to promote the oxidation reaction and serve as a cosolvent for the oxidant. The volume amount of the tert-butyl alcohol is generally 0.5 to 2 mL / g based on the mass of Dess-Martin reagent (DMP).

[0025] In the step (1), after the selective oxidation reaction is completed, the reaction solution is post-treated to obtain 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one shown in Formula 1, the intermediate product shown in Formula 5, or the intermediate product shown in Formula 7;

[0026] The post-treatment method is generally as follows: washing the reaction solution with water, washing with saturated brine, drying, filtering, concentrating, and separating by column chromatography to obtain 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one shown in Formula 1, the intermediate product shown in Formula 5, or the intermediate product shown in Formula 7;

[0027] (2) 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one represented by Formula 1, the intermediate product represented by Formula 5, or the intermediate product represented by Formula 7 is subjected to a condensation reaction with an aldehyde R-CHO in an alcoholic solution of potassium hydroxide to prepare a ginsenoside derivative represented by Formula I, II, or III, respectively;

[0028] The molar ratio of 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one represented by formula 1, the intermediate product represented by formula 5 or the intermediate product represented by formula 7 and the aldehyde R-CHO is 1:0.1-10, preferably 1:1.5-5.

[0029] The reaction temperature is room temperature, and the reaction time is 1 to 8 hours.

[0030] Furthermore, the alcohol in the alcoholic solution of potassium hydroxide is methanol or ethanol, wherein the mass percentage concentration of potassium hydroxide is 1% to 50%, preferably 40% to 45%;

[0031] The volume usage of the alcoholic potassium hydroxide solution is 10 to 50 mL / g, preferably 20 to 40 mL / g, based on the mass of 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one represented by Formula 1, the intermediate product represented by Formula 5, or the intermediate product represented by Formula 7.

[0032] The molar ratio of the 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one represented by Formula 1, the intermediate product represented by Formula 5 or the intermediate product represented by Formula 7 and potassium hydroxide is 1:5-20, preferably 1:8-15.

[0033] In the step (2), after the condensation reaction is completed, the reaction solution is post-treated to prepare the ginsenoside derivatives represented by formula I, II or III respectively; the post-treatment steps are generally: the reaction solution is diluted with ethyl acetate, then washed with water and saturated brine, dried and then filtered, concentrated, and separated by column chromatography to prepare the ginsenoside derivatives represented by formula I, II or III.

[0034] (3) The ginsenoside derivatives represented by formula I, II or III are subjected to a reduction reaction in an alcoholic solution of NaBH4 to obtain ginsenoside derivatives represented by formula IV, V or VI, respectively;

[0035] The molar ratio of the ginsenoside derivative represented by formula I, II or III and NaBH4 is 1:1-10, preferably 1:1.5-5.

[0036] The reaction temperature is room temperature, and the reaction time is 0.1 to 8 hours.

[0037] In the alcohol solution of NaBH4, the alcohol solvent is ethanol, methanol or isopropanol.

[0038] The volume usage of the NaBH4 alcohol solution is 10 to 50 mL / g, preferably 20 to 40 mL / g, based on the mass of the ginsenoside derivative represented by Formula I, II or III.

[0039] In the step (3), after the reduction reaction is completed, the reaction solution is post-treated to prepare the ginsenoside derivatives represented by formula IV, V or VI respectively; the post-treatment steps are generally as follows: the reaction solution is diluted with ethyl acetate, then washed with water and saturated brine, dried, filtered, concentrated, and separated by column chromatography to prepare the ginsenoside derivatives represented by formula I, II or III.

[0040] The pharmaceutically acceptable salts of the derivatives of the present invention refer to conventional acid addition salts, which have the same pharmaceutical efficacy as the derivatives and are salts formed with suitable non-toxic organic or inorganic acids.

[0041] The present invention also provides ginsenoside derivatives represented by formula I, II, III, IV, V or VI and pharmaceutically acceptable salts thereof, which have anti-inflammatory activity and can be used to prepare anti-inflammatory drugs or anti-inflammatory pharmaceutical compositions; specifically, they can be used to prepare drugs or pharmaceutical compositions for treating and preventing diseases related to inflammatory bowel disease, sepsis, etc.

[0042] The ginsenoside derivatives of the present invention or pharmaceutically acceptable salts thereof can be added with pharmaceutically acceptable carriers to prepare common pharmaceutical preparations, such as tablets, capsules, powders, syrups, liquids, suspensions, injections, etc., and can also be added with common pharmaceutical excipients acceptable to the human body, such as flavorings, sweeteners, liquid or solid fillers or diluents.

[0043] The ginsenoside derivatives of the present invention can be administered clinically by oral administration, injection, or the like.

[0044] The clinical dosage of the ginsenoside derivatives of the present invention is 0.01 mg to 1000 mg / day, and may deviate from this range depending on the severity of the disease or the dosage form.

[0045] Compared with the prior art, the present invention has the following advantages:

[0046] 1. The compounds exhibit excellent anti-inflammatory activity. At a concentration of 20 μM, the compounds in this series exhibited stronger inhibitory effects on NO production than the commercially available drug hydrocortisone sodium succinate, demonstrating superior anti-inflammatory activity. Furthermore, the anti-inflammatory activity is superior to that of esterified derivatives disclosed in the prior art (e.g., Compound 8 disclosed in Patent CN 111647036A).

[0047] 2. The compounds have lower cytotoxicity: at a concentration of 20 μM, the cell viability rate reached over 95%. Most of the compounds had higher cell viability in the experiment than the commercially available drug hydrocortisone sodium succinate.

[0048] 3. The compound structure is more stable. Compared with the esterified derivatives with anti-inflammatory activity disclosed in the prior art, the derivatives of the present invention do not contain ester bonds and are therefore less susceptible to hydrolysis by esterases in plasma, thereby having greater plasma stability and improving drug bioavailability.

[0049] In summary, the ginsenoside derivatives provided by the present invention have better effects on inhibiting LPS-induced NO release than existing clinical drugs, and have good safety, lower toxicity and stronger stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a graph showing the anti-inflammatory activity test results of ginsenoside derivatives.

[0051] Figure 2 This is a graph showing the cytotoxicity test results of ginsenoside derivatives.

[0052] Figure 3 The MitoSOX experiment was detected by enzyme-labeled instrument, and the NLRP3 experiment was detected by Western Blot. The left picture shows the MitoSOX experiment results, and the right picture shows the NLRP3 experiment results. DETAILED DESCRIPTION

[0053] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0054] Example 1: 20(S)-12β,20-dihydroxy-2-(4-nitrobenzylidene)-dammarane-24-ene-3-one (I-1)

[0055] 20S-protopanaxadiol (50 mg) was dissolved in dichloromethane (1 mL), and NaHCO₃ (50 mg) was added. DMP (55 mg) was added, and tert-butanol (40 μL) was added dropwise. The mixture was stirred at room temperature for 6 h. The mixture was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the intermediate product 1 [20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one] in an 80% yield.

[0056] Intermediate product 1 (30 mg) was dissolved in a KOH ethanol solution (KOH mass concentration 40%, 1 mL), 4-nitrobenzaldehyde (30 mg) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the final product I-1 in a yield of 91%. 1 HNMR(400MHz, CDCl3)δ8.25(d,J=8.5Hz,2H),7.55(d,J=8.5Hz,2H),7.46(s,1H) ,5.16(t,J=7.0Hz,1H),4.04(s,1H),3.68(td,J=9.8,4.6Hz,1H),3.05(d,J=16.2 Hz,1H),,2.31(d,J=16.2Hz,1H),1.78(t,J=10.5Hz,1H),1.69(s,3H),1.63(s,3H ),1.23(s,3H),1.19(s,3H),1.15(s,3H),1.03(s,3H),0.94(s,3H),0.86(s,3H).

[0057] Example 2: 20(S)-12β,20-dihydroxy-2-(4-cyanobenzylidene)-dammarane-24-ene-3-one (I-2)

[0058] Intermediate product 1 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-cyanobenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product, product I-2, in a yield of 71%. 1 HNMR(400MHz, CDCl3) δ7.69(d,J=7.9Hz,2H),7.49(d,J=7.9Hz,2H),7.41(s,1 H),5.17(t,J=6.4Hz,1H),3.67(td,J=9.8,4.6Hz,1H),3.03(d,J=16.2Hz,1H) ,2.30(d,J=15.7Hz,1H),1.78(t,J=10.5Hz,1H),1.70(s,3H),1.64(s,3H),1. 23(s,3H),1.18(s,3H),1.14(s,3H),,1.03(s,3H),0.94(s,3H),0.86(s,3H).

[0059] Example 3: 20(S)-12β,20-dihydroxy-2-(4-trifluoromethylbenzylidene)-dammarane-24-ene-3-one (I-3)

[0060] Intermediate product 1 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-trifluoromethylbenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product I-3 in a 70% yield. 1 HNMR(400MHz, CDCl3)7.65(d,J=8.2Hz,2H),7.50(d,J=8.0Hz,2H),7.46(s,1H), 5.17(t,J=7.1Hz,1H),3.97(s,2H),3.67(td,J=9.8,4.6Hz,1H),3.06(d,J=16.2 Hz,1H),2.29(d,J=16.1Hz,1H),1.78(t,J=10.5Hz,1H),1.69(s,3H),1.63(s,3H ),1.22(s,3H),1.18(s,3H),1.15(s,3H),1.02(s,3H),0.93(s,3H),0.85(s,3H).

[0061] Example 4: 20(S)-12β,20-dihydroxy-2-(4-bromobenzylidene)-dammarane-24-ene-3-one (I-4)

[0062] Intermediate product 1 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-bromobenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product I-4 in a yield of 74%. 1 HNMR(400MHz, CDCl3)δ7.52(d,J=8.5Hz,2H),7.39(s,1H),7.28(d,J=8.1Hz,2H) ,5.18(t,J=7.1Hz,1H),4.04(s,2H),3.68(td,J=10.4,5.7Hz,1H),3.03(d,J=17. 3Hz,1H),2.26(d,J=16.1Hz,1H),1.78(t,J=10.5Hz,1H),1.69(s,3H),1.64(s,3H ),1.22(s,3H),1.17(s,3H),1.13(s,3H),1.01(s,3H),0.93(s,3H),0.82(s,3H).

[0063] Example 5: 20(S)-12β,20-dihydroxy-2-(4-methylbenzylidene)-dammarane-24-ene-3-one (I-5)

[0064] Intermediate product 1 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-methylbenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product, product I-5, in a yield of 66%. 1 HNMR (400MHz, CDCl3) δ7.46 (s, 1H), 7.33 (d, J = 7.5Hz, 2H), 7.21 (d, J = 7.5Hz, 2 H),5.17(t,J=7.1Hz,1H),4.06(s,2H),3.71(td,J=10.4,5.7Hz,1H),3.09(d,J =16.1Hz,1H),2.36(s,3H),2.30(d,J=15.1Hz,1H),1.69(s,3H),1.64(s,3H), 1.23(s,3H),1.17(s,3H),1.12(s,3H),1.03(s,3H),0.94(s,3H),0.85(s,3H).

[0065] Example 6: 20(S)-12β,20-dihydroxy-2-(3-pyridylmethylene)-dammarane-24-ene-3-one (I-6)

[0066] Intermediate product 1 (40 mg) was dissolved in a 40% ethanolic solution of KOH (1 mL). 2-pyridinecarboxaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product, product I-6, in an 80% yield. 1 HNMR(400MHz, CDCl3)δ8.71–8.64(m,1H),8.55–8.47(m,1H),,7.75(d,J=7.9Hz,1H), 7.40(s,1H),7.35(dd,J=7.6,4.7Hz,1H),5.14(t,J=7.1Hz,1H),3.67(td,J=10.3,4.8 Hz,1H),3.07(d,J=16.2Hz,1H),2.32(d,J=16.5Hz,1H),1.80(t,J=10.6Hz,1H),1.68( s,3H),1.62(s,3H),1.19(s,6H),1.14(s,3H),1.04(s,3H),0.93(s,3H),0.88(s,3H).

[0067] Example 7: 20(S)-12β,20-dihydroxy-2-benzylidene-dammarane-24-ene-3-one (I-7)

[0068] Intermediate product 1 (40 mg) was dissolved in a 40% ethanolic solution of KOH (1 mL), and benzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product, product I-7, in an 82% yield. 1 H NMR (400MHz, CDCl3) δ7.48–7.36(m,5H),7.34–7.29(m,1H),5.17(t,J=7.1Hz,1H),3.67(td,J=10.4,5.0Hz,1H),3.11(d,J=17.6Hz,1H),2.31(d, J=14.8Hz,1H),1.78(t,J=10.6Hz,1H),1.69(s,3H),1.64(s,3H),1.22( s,3H),1.18(s,3H),1.13(s,3H),1.03(s,3H),0.93(s,3H),0.86(s,3H).

[0069] Example 8: 20(S)-12β,20-dihydroxy-2-cyclohexylidene-dammarane-24-ene-3-one (I-8)

[0070] Intermediate product 1 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL). Cyclohexylcarboxaldehyde (40 mg) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the final product, product I-8, in a yield of 79%. 1 HNMR (400MHz, CDCl3) δ6.45(d,J=9.9Hz,1H),5.17(t,J=8.2Hz,1H),3.66(td,J=10.5,4.9Hz,1H),2.78(d,J=15.6 Hz,1H),1.71(s,3H),1.65(s,3H),1.23(s,3H),1.09(s,3H),1.07(s,3H),1.04(s,3H),0.93(s,3H),0.84(s,3H).

[0071] Example 9: (20S,24R)-Epoxy-12β,25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one (II-1) 20S-protopanaxadiol (2.0 g) was dissolved in dichloromethane (50 mL), and m-CPBA (1.0 g) was added. The mixture was stirred at room temperature for 3 h. The mixture was diluted with chloroform, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain intermediates 3 [(20S,24R)-epoxydammarane-3β,12β,25-triol] and 4 [(20S,24S)-epoxydammarane-3β,12β,25-triol].

[0072] Intermediate product 3 (1 g) was dissolved in dichloromethane (20 mL), and NaHCO3 (528 mg) was added. DMP (1 g) was added, and tert-butanol (1 mL) was added dropwise. The mixture was stirred at room temperature for 6 h. The mixture was diluted with chloroform, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain intermediate product 5 [(20S,24R)-epoxy-12β,25-dihydroxy-dammarane-3-one] in an 83% yield.

[0073] Intermediate product 5 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL). 4-nitrobenzaldehyde (40 mg) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-1, in a 65% yield. 1HNMR (400MHz, CDCl3) δ8.25(d,J=8.8Hz,2H),7.53(d,J=8.6Hz,2H),7.45(d,J=2.4Hz,1H),3.86(dd,J=10.9,5.3Hz,1H),3.61(td,J=10.4,4.6 Hz,1H),3.05(dd,J=16.5,1.8Hz,1H),1.29(s,3H),1.22(s,3H),1.19(s ,3H),1.15(s,3H),1.10(s,3H),1.06(s,3H),0.96(s,3H),0.87(s,3H).

[0074] Example 10: (20S,24R)-epoxy-12β,25-dihydroxy-2-(4-cyanobenzylidene)-dammarane-3-one (II-2)

[0075] Intermediate product 5 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 ml), and 4-cyanobenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-2, in a yield of 55%. 1 HNMR (400MHz, CDCl3) δ7.67(d,J=8.4Hz,2H),7.48(d,J=8.1Hz,2H),7.39(t,J=2.2Hz,1H),3.87(dd,J=10.9,5.3Hz,1H),3.61(td,J=10.3,4.5 Hz,1H),3.04(dd,J=16.5,1.8Hz,1H),1.30(s,3H),1.23(s,3H),1.19(s ,3H),1.14(s,3H),1.10(s,3H),1.06(s,3H),0.96(s,3H),0.87(s,3H).

[0076] Example 11: (20S,24R)-epoxy-12β,25-dihydroxy-2-(4-trifluoromethylbenzylidene)-dammarane-3-one (II-3)

[0077] Intermediate product 5 (40 mg) was dissolved in a KOH ethanol solution (KOH mass concentration 40%, 1 ml), 4-trifluoromethylbenzaldehyde (40 mg) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-3, in a yield of 75%. 1HNMR (400MHz, CDCl3) δ7.64(d,J=8.2Hz,2H),7.48(d,J=8.4Hz,2H),7.45(s,1H),3.86(dd,J=10.9,5.4Hz,1H),3.60(td,J=10.4,4.6Hz,1 H),3.07(dd,J=16.5,1.7Hz,1H),1.29(s,3H),1.23(s,3H),1.19(s,3H),1.15(s,3H),1.10(s,3H),1.06(s,3H),0.96(s,3H),0.87(s,3H).

[0078] Example 12: (20S,24R)-epoxy-12β,25-dihydroxy-2-(4-bromobenzylidene)-dammarane-3-one (II-4)

[0079] Intermediate product 5 (40 mg) was dissolved in KOH ethanol solution (KOH mass concentration 40%, 1 ml), 4-bromobenzaldehyde (40 mg) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-4, in a yield of 63%. 1 HNMR (400MHz, CDCl3) δ7.51(d,J=8.5Hz,2H),7.37(t,J=2.1Hz,1H),7.27(d,J=8.6Hz,2H),5.89(s,1H),3.87(dd,J=10.8,5.4Hz,1H),3.61(td,J=10. 4,4.6Hz,1H),3.04(dd,J=16.4,1.8Hz,1H),1.29(s,3H),1.24(s,3H),1.1 8(s,3H),1.13(s,3H),1.11(s,3H),1.06(s,3H),0.96(s,3H),0.86(s,3H).

[0080] Example 13: (20S,24R)-epoxy-12β,25-dihydroxy-2-(4-methylbenzylidene)-dammarane-3-one (II-5)

[0081] Intermediate product 5 (40 mg) was dissolved in a 40% ethanolic solution of KOH (1 ml), and 4-methylbenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-5, in a yield of 86%. 1HNMR(400MHz, CDCl3)7.44(s,1H),7.31(d,J=8.2Hz,2H),7.19(d,J=7.9Hz,2H),3.88(dd,J=10.8,5.3Hz,1H),3.62(td,J=10.3,4.5Hz,1H),3.0 9(dd,J=16.5,1.8Hz,1H),2.37(s,3H),1.29(s,3H),1.24(s,3H),1.18( s,3H),1.12(s,3H),1.11(s,3H),1.06(s,3H),0.96(s,3H),0.87(s,3H).

[0082] Example 14: (20S,24R)-epoxy-12β,25-dihydroxy-2-(3-pyridylmethylene)-dammarane-3-one (II-6)

[0083] Intermediate product 5 (40 mg) was dissolved in a 40% ethanolic solution of KOH (1 mL). 2-pyridinecarboxaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-6, in a 64% yield. 1 H NMR (400MHz, CDCl3) δ8.68–8.62(m,1H),8.57–8.52(m,1H),7.74(d,J=8.0Hz,1H), 7.41(s,1H),7.34(dd,J=8.0,4.6Hz,1H),5.84(s,1H),3.87(dd,J=10.9,5.4Hz,1H) ,3.60(td,J=10.4,4.7Hz,1H),3.05(dd,J=16.5,1.8Hz,1H),1.29(s,3H),1.23(s, 3H),1.19(s,3H),1.14(s,3H),1.10(s,3H),1.06(s,3H),0.96(s,3H),0.88(s,3H).

[0084] Example 15: (20S,24R)-epoxy-12β,25-dihydroxy-2-benzylidene-dammarane-3-one (II-7)

[0085] Intermediate product 5 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL). Benzaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-7, in a 63% yield. 1H NMR (400MHz, CDCl3) δ7.46–7.36(m,5H),7.34–7.29(m,1H),5.87(s,1H),3.87(dd,J=10.8,5.3Hz,1H),3.62(td,J=10.4,4.6Hz,1H),3 .11(dd,J=16.5,1.8Hz,1H),1.29(s,3H),1.24(s,3H),1.19(s,3H),1.13(s,3H),1.10(s,3H),1.06(s,3H),0.96(s,3H),0.88(s,3H).

[0086] Example 16: (20S,24R)-Epoxy-12β,25-dihydroxy-2-cyclohexylidene-dammarane-3-one (II-8)

[0087] Intermediate product 5 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL). Cyclohexylcarboxaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product II-8, in a 63% yield. 1 H NMR (400MHz, CDCl3) δ6.46 (ddd, J=9.9, 2.8, 1.4Hz, 1H), 3.90 (dd, J=10.8, 5.4Hz, 1H), 3.60 (td, J=10.3, 4.5Hz, 1H), 2.81 (dd, J=15.7,1.5Hz,1H),1.30(s,3H),1.25(s,3H),1.12(s,3H),1.09(s,3H),1.08(s,3H),1.06(s,3H),0.95(s,3H),0.85(s,3H).

[0088] Example 17: (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one (III-1)

[0089] Intermediate product 4 (330 mg) was dissolved in dichloromethane (7 mL), and NaHCO₃ (174 mg) was added. DMP (352 mg) was added, and tert-butanol (300 μL) was added dropwise. The mixture was stirred at room temperature for 6 h. The mixture was diluted with chloroform, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain intermediate product 5 [(20S,24S)-epoxy-12β,25-dihydroxy-dammarane-3-one] in an 81% yield.

[0090] Intermediate product 7 (26 mg) was dissolved in a 40% ethanolic solution of KOH (1 mL). 4-nitrobenzaldehyde (12 mg) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product III-1, in a 64% yield. 1 HNMR (400MHz, CDCl3) δ8.26 (d, J=8.8Hz, 2H), 7.53 (d, J=8.6Hz, 2H), 7.45 (dd, J= 2.9,1.6Hz,1H),3.86(dd,J=8.7,6.6Hz,1H),3.60(td,J=10.5,4.4Hz,1H),3.02 (dd,J=16.5,1.8Hz,1H),2.31(dd,J=16.7,2.2Hz,1H),1.28(s,3H),1.27(s,3H) ,1.19(s,3H),1.14(s,3H),1.09(s,3H),1.03(s,3H),0.95(s,3H),0.84(s,3H).

[0091] Example 18: (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-cyanobenzylidene)-dammarane-3-one (III-2)

[0092] Intermediate product 7 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-cyanobenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product III-2 in a 67% yield. 1 HNMR (400MHz, CDCl3) δ7.69(d,J=8.4Hz,2H),7.48(d,J=8.3Hz,2H),7.39(t,J=2.2Hz,1H),3.87(dd,J=8.7,6.6Hz,1H),3.59(td,J=10.5,4.4H z,1H),3.00(dd,J=16.6,1.8Hz,1H),1.29(s,3H),1.28(s,3H),1.18(s ,3H),1.13(s,3H),1.09(s,3H),1.03(s,3H),0.94(s,3H),0.84(s,3H).

[0093] Example 19: (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-trifluoromethylbenzylidene)-dammarane-3-one (III-3)

[0094] Intermediate product 7 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-trifluoromethylbenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product III-3 in an 82% yield. 1 HNMR (400MHz, CDCl3) δ7.68–7.62(m,2H),7.51–7.47(m,2H),7.44(t,J=2.1Hz,1H),3 .86(dd,J=8.7,6.6Hz,1H),3.60(td,J=10.5,4.5Hz,1H),3.04(dd,J=16.5,1.8Hz,1H) ,2.31(dd,J=17.3,2.4Hz,1H),2.23(ddd,J=10.9,9.2,3.7Hz,1H),1.28(s,3H),1.28( s,3H),1.18(s,3H),1.14(s,3H),1.09(s,3H),1.03(s,3H),0.94(s,3H),0.84(s,3H).

[0095] Example 20: (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-bromobenzylidene)-dammarane-3-one (III-4)

[0096] Intermediate product 7 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-bromobenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product III-4 in a 55% yield. 1 HNMR (400MHz, CDCl3) δ7.53(d,J=8.5Hz,2H),7.38–7.34(m,1H),7.27(d,J=8.4Hz,2H),3.86(dd,J=8.7,6.6Hz,1H),3.60(td,J=10.5, 4.5Hz,1H),3.01(dd,J=16.4,1.8Hz,1H),1.28(s,6H),1.17(s,3H),1.12(s,3H),1.09(s,3H),1.03(s,3H),0.94(s,3H),0.83(s,3H).

[0097] Example 21: (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-methylbenzylidene)-dammarane-3-one (III-5)

[0098] Intermediate product 7 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL), and 4-methylbenzaldehyde (40 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product III-5 in a 90% yield. 1 HNMR(400MHz, CDCl3)δ7.43(t,J=2.3Hz,1H),7.32(d,J=8.2Hz,2H),7.20(d,J=8.0Hz, 2H),3.86(dd,J=8.8,6.7Hz,1H),3.61(td,J=10.5,4.5Hz,1H),3.06(dd,J=16.4,1.7H z,1H),2.38(s,3H),2.30(d,J=16.2Hz,1H),2.23(ddd,J=10.9,9.1,3.7Hz,1H),1.28( s,6H),1.17(s,3H),1.11(s,3H),1.09(s,3H),1.03(s,3H),0.94(s,3H),0.84(s,3H).

[0099] Example 22: (20S,24S)-epoxy-12β,25-dihydroxy-2-(3-pyridylmethylene)-dammarane-3-one (III-6)

[0100] Intermediate product 7 (40 mg) was dissolved in a 40% ethanolic solution of KOH (1 mL). 2-pyridinecarboxaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product III-6, in a 93% yield. 1 H NMR (400MHz, CDCl3) δ8.70–8.61(m,1H),8.61–8,50(m,1H),7.74(d,J=8.0Hz,1H),7.40(s,1H) ,7.36(dd,J=8.0,4.8Hz,1H),3.86(dd,J=8.7,6.6Hz,1H),3.59(td,J=10.5,4.5Hz,1H),3.02( dd,J=16.5,1.8Hz,1H),2.34(d,J=16.5Hz,1H),2.23(ddd,J=11.0,9.2,3.8Hz,1H),1.28(s,3H ),1.27(s,3H),1.19(s,3H),1.13(s,3H),1.09(s,3H),1.03(s,3H),0.95(s,3H),0.85(s,3H).

[0101] Example 23: (20S,24S)-Epoxy-12β,25-dihydroxy-2-benzylidene-dammarane-3-one (III-7)

[0102] Intermediate product 7 (40 mg) was dissolved in a 40% ethanolic solution of KOH (1 mL). Benzaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product III-7 in a 71% yield. 1 H NMR(400MHz, CDCl3)δ7.43(d,J=1.1Hz,1H),7.42–7.38(m,4H),7.36–7.30(m,1H),3 .86(dd,J=8.8,6.8Hz,1H),3.60(td,J=10.5,4.5Hz,1H),3.07(dd,J=16.1,1.5Hz,1 H),2.32(d,J=16.3Hz,1H),2.23(ddd,J=10.9,9.2,3.8Hz,1H),1.28(s,3H),1.28(s ,3H),1.18(s,3H),1.12(s,3H),1.09(s,3H),1.03(s,3H),0.94(s,3H),0.85(s,3H).

[0103] Example 24: (20S,24S)-Epoxy-12β,25-dihydroxy-2-cyclohexylidene-dammarane-3-one (III-8)

[0104] Intermediate product 7 (40 mg) was dissolved in a 40% ethanol solution of KOH (1 mL). Cyclohexylcarboxaldehyde (40 μL) was added and stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product, product III-8, in a yield of 79%. 1 H NMR (400MHz, CDCl3) δ6.45(ddd,J=9.9,2.8,1.4Hz,1H),3.87(dd,J=8.8,6.7Hz,1H),3.59(td,J=10.4,4.4Hz,1H),2.77(dd, J=15.8,1.6Hz,1H),1.30(s,3H),1.29(s,3H),1.11(s,3H),1.08(s,3H),1.07(s,3H),1.03(s,3H),0.94(s,3H),0.82(s,3H).

[0105] Example 25: 20(S)-dammarane-2-(4-nitrobenzylidene)-24-ene-3β-12β,20-triol (IV-1)

[0106] Derivative I-1 (40 mg) was dissolved in isopropanol (1 mL), and NaBH4 (10 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product IV-1 in a yield of 70%. 1 HNMR (400MHz, CDCl3) δ8.15(d,J=8.8Hz,2H),7.32(d,J=8.2Hz,2H),6.79(s,1H),5.14(t,J=7.8Hz,1H),3.90(s,1H),3.57(td,J=10.4,5. 1Hz,1H),2.95(d,J=12.8Hz,1H),1.68(s,3H),1.62(s,3H),1.19(s,3H),1.15(s,3H),0.94(s,3H),0.90(s,3H),0.77(s,3H),0.73(s,3H).

[0107] Example 26: 20(S)-dammarane-2-(4-cyanobenzylidene)-24-ene-3β-12β,20-triol (IV-2)

[0108] Derivative I-2 (40 mg) was dissolved in isopropanol (1 mL), and NaBH4 (10 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product IV-2 in a yield of 75%. 1 HNMR (400MHz, CDCl3) δ7.55(d,J=7.9Hz,2H),7.28(d,J=8.1Hz,2H),6.74(s,1H),5.14(t,J=7.1Hz,1H),3.88(d,J=2.0Hz,1H),3.57(td,J=10. 4,5.1Hz,1H),2.93(d,J=12.6Hz,1H),1.68(s,3H),1.62(s,3H),1.19(s ,3H),1.14(s,3H),0.94(s,3H),0.90(s,3H),0.76(s,3H),0.72(s,3H).

[0109] Example 27: (20S,24R)-Epoxydamane-2-(4-nitrobenzylidene)-3β,12β,25-triol (V-1)

[0110] Derivative II-1 (40 mg) was dissolved in isopropanol (1 mL), and NaBH4 (10 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product V-1 in a yield of 69%. 1 H NMR (400MHz, CDCl3) δ8.20(d,J=8.8Hz,2H),7.32(d,J=8.4Hz,2H),6.78(s,1H),3 .88(d,J=2.0Hz,1H),3.83(dd,J=9.0,6.6Hz,1H),3.50(td,J=10.5,4.5Hz,1H),2 .90(d,J=12.9Hz,1H),2.18(ddd,J=10.9,9.1,3.6Hz,1H),1.26(s,3H),1.26(s,3 H),1.14(s,3H),1.07(s,3H),0.94(s,3H),0.91(s,3H),0.76(s,3H),0.69(s,3H).

[0111] Example 28: (20S, 24R)-Epoxydammarane-2-(3-pyridylmethylene)-3β, 12β, 25-triol (V-6)

[0112] Derivative II-6 (40 mg) was dissolved in isopropanol (1 mL), and NaBH4 (10 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product V-6 in a yield of 73%. 1 H NMR(400MHz, CDCl3) δ8.44–8.40(m,2H),7.52(td,J=7.8,2.0Hz,1H),6.71(s,1 H),3.87(d,J=2.1Hz,1H),3.83(dd,J=8.9,6.6Hz,1H),3.50(td,J=10.5,4.6Hz ,1H),2.88(d,J=12.9Hz,1H),2.18(ddd,J=10.9,9.2,3.7Hz,1H),1.26(s,6H), 1.13(s,3H),1.08(s,3H),0.94(s,3H),0.91(s,3H),0.76(s,3H),0.72(s,3H).

[0113] Example 29: (20S,24S)-Epoxydamane-2-(4-nitrobenzylidene)-3β,12β,25-triol (VI-1)

[0114] Derivative III-1 (40 mg) was dissolved in isopropanol (1 mL), and NaBH4 (10 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the final product VI-1 in a yield of 78%. 1 H NMR (400MHz, CDCl3) δ8.16 (d, J=8.8Hz, 2H), 7.35–7.29 (m, 2H), 6.79 (s, 1H), 5.8 1(s,1H),4.81(s,1H),3.83(dd,J=10.9,5.3Hz,1H),3.51(td,J=10.3,4.7Hz,1H ),2.93(d,J=12.9Hz,1H),2.24(td,J=10.6,4.5Hz,1H),1.26(s,3H),1.20(s,3H ),1.14(s,3H),1.08(s,3H),0.96(s,3H),0.93(s,3H),0.77(s,3H),0.71(s,3H).

[0115] Example 30: Detection of the inhibitory activity of ginsenoside derivatives on NO production;

[0116] The present invention uses the Griess test to detect the NO release level induced by LPS (LPS, Lipopolysaccharide), and then evaluates the anti-inflammatory activity of ginsenoside derivatives.

[0117] RAW264.7 cells in the logarithmic growth phase were taken and the concentration was 5×10 4 Each well was seeded in a 96-well plate and incubated for 24 hours. The cells were then stimulated with LPS (1 μg / mL) to establish a model. A 20 μM concentration of a ginsenoside derivative and the positive drug hydrocortisone (HSS) were also added. The 96-well plate was then placed in an incubator and incubated for another 24 hours. After incubation, the plate was removed and the supernatant from each well was aseptically aspirated into another 96-well plate, with 100 μL aliquoted per well. Nitrite levels in the aspirated supernatant were determined using Griess reagent to determine NO content. The absorbance at 540 nm (OD540) was then measured in a microplate reader (SpectraMax M3). The blank group consisted of a group without LPS or drug treatment, while the control group consisted of a group stimulated with LPS but without compound treatment. LPS-induced NO release in RAW264.7 cells was measured using Griess reagent.

[0118] NO inhibition rate = [control group (OD540) - compound (OD540)] / [control group (OD540) - blank group (OD540)] × 100%

[0119] The biological activity test results of the ginsenoside derivatives of the present invention are as follows: Figure 1 shown.

[0120] The legend is as follows:

[0121] Each value is the mean ± SD from three parallel experiments (n = 3);

[0122] Compared with the blank group, #p<0.05,##p<0.01,###p<0.001;

[0123] Compared with the LPS stimulation group *p<0.05, **p<0.01, ***p<0.001;

[0124] Figure 1 The vertical axis represents the NO concentration.

[0125] Figure 1 The results showed that

[0126] The ginsenoside derivatives provided by the present invention have good anti-inflammatory activity, all of which significantly inhibit the increase in NO release induced by LPS, and the anti-inflammatory activity is superior to that of a positive drug (hydrocortisone); the inhibition rate of compounds I-1, I-2, I-6, II-1, II-2, II-6, III-1, III-2, IV-1, IV-2, and V-1 on NO production at a concentration of 20 μM is more than twice that of the positive control hydrocortisone sodium succinate, and a high inhibition rate on NO production is still achieved at a concentration of 10 μM.

[0127] Example 31: MTT assay for in vitro cytotoxicity of ginsenoside derivatives

[0128] The MTT assay, also known as the MTT colorimetric method, is a method for measuring cell viability and growth. Its principle is that succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water-insoluble, blue-purple crystalline formazan, which then deposits within the cells. This phenomenon does not occur in dead cells. Formazan crystals can be dissolved in DMSO, and their absorbance measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA). Within a certain cell count range, the absorbance value is proportional to the number of viable cells, thus indirectly reflecting the number of viable cells.

[0129] This experiment was carried out together with Example 30.

[0130] MTT assay: Add 20 μL / well of 5 mg / mL MTT solution to cells treated with 20 μM ginsenoside derivatives from Example 30 and incubate in an incubator for 4 h. Discard the supernatant, add 150 μL / well of DMSO, and mix thoroughly by vortexing. Then, measure the absorbance of the samples at 570 nm (OD570) using a SpectraMax M3.

[0131] Cell survival rate = [1-survival rate of experimental group] / average survival rate of control group × 100%

[0132] The cytotoxicity test results of the ginsenoside derivatives of the present invention are as follows: Figure 2 .

[0133] The legend is as follows:

[0134] Each value is the mean ± SD from three parallel experiments (n = 3);

[0135] Different letters indicate significant differences among the data groups (p<0.05);

[0136] Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001.

[0137] The results of Example 31 show that

[0138] Compared with the model group cells, the ginsenoside derivatives provided by the present invention showed no downward trend in cell survival rate, indicating that the ginsenoside derivatives were non-cytotoxic at a concentration of 20 μM, and their cell survival rates generally met or exceeded the standard of low toxicity. The cytotoxicity of most ginsenoside derivatives was lower than that of the clinical drug hydrocortisone. Compounds I-1, I-2, I-6, II-1, II-2, II-6, III-1, III-2, IV-1, IV-2, and V-1 exhibited strong NO inhibition effects while also exhibiting low cytotoxicity at 20 μM.

[0139] Example 32: Microplate reader detection of MitoSOX experiment

[0140] Take RAW264.7 cells in the logarithmic growth phase and adjust the cell density to 5×10 5 / mL, 100 μL of cell suspension per well was plated onto a black 96-well plate and incubated in an incubator for 4 hours to ensure cell attachment. The control group remained untreated, while the model group received an equal volume of DMSO. The HSS group (20 μM) and the drug-treated group received I-1 (5 μM, 10 μM, and 20 μM) for 2 hours. LPS (1 μg / mL) was then added and incubated in an incubator for another 24 hours and 30 minutes. The 96-well plate was removed and, under sterile conditions, the supernatant was discarded. The plate was then washed twice with 37°C preheated PBS (100 μL / well). Then, 50 μL of a 5 μM mitoSOX probe was added and the plate was incubated in the dark for 15 minutes. The glass-bottomed dish was removed and, under sterile conditions, the mitoSOX probe was discarded. The plate was washed twice with 37°C preheated PBS (100 μL / well). Finally, 50 μL of PBS was added and fluorescence intensity was measured using a microplate reader at an excitation wavelength of 540 nm and an emission wavelength of 570 nm.

[0141] Western Blot detection of NLRP3

[0142] Take cells in the logarithmic growth phase and adjust the cell density to 1×10 7 / mL, 2mL of cell suspension per well was added to a 6-well plate and incubated in an incubator for 24 hours. The control group was untreated, while the model group was treated with an equal volume of DMSO, the HSS group (20μM), and the drug-treated group I-1 (5μM, 10μM, and 20μM) for 2 hours. LPS (1μg / mL) was then added and incubated in an incubator for another 24 hours. The cells were lysed to detect NLRP3 protein expression.

[0143] The legend is as follows:

[0144] Each value is the mean ± SD from three parallel experiments (n = 3);

[0145] Different letters indicate significant differences among the data groups (p<0.05);

[0146] Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001.

[0147] The results of Example 32 showed that LPS treatment primarily induced the production of MtROS. At 0.5 and 24 hours of treatment, I-1 almost completely inhibited LPS-induced MtROS production, demonstrating greater efficiency than HSS. These data suggest that the inhibition of LPS-triggered oxidative stress is closely related to the activity of I-1.

[0148] In this study, 24-hour LPS treatment triggered a significant upregulation of NLRP3 protein. I-1, but not HSS, significantly inhibited this upregulation in a dose-dependent manner. These results are consistent with those from experiments on MtROS generation. NLRP3 inflammasome activation may also be closely linked to LPS-triggered NO production, and NLRP3 protein expression is essential for LPS-triggered iNOS upregulation in RAW264.7 cells. Our data suggest that I-1 primarily blocks NLRP3 upregulation by inhibiting LPS-induced MtROS, thereby suppressing the release of NO and other inflammatory factors.

[0149] Example 33: Plasma stability experiment

[0150] Preparation of plasma working solution: Draw 4 mL of fresh rat blood into a tube containing sodium heparin and store on ice at 4°C. Centrifuge at 5000 × g. Store the supernatant at -80°C.

[0151] Prepare the working solution of the drug: take 20 μL of pH 7.4 phosphate buffer and add 80 μL of the compound I-1 storage solution (10 mM).

[0152] Drug stability test in plasma: Mix the plasma working solution and the drug working solution, take 50 μL to 350 μL of methanol, mix well, take the supernatant, pass through a microporous filter membrane, and store at -80°C. This sample is recorded as time point 0.

[0153] The remaining pooled plasma was divided into six 50 μL aliquots. Incubate at 37°C. At 0.5, 1, 2, 4, 8, and 24 h, 350 μL of methanol was added, mixed, and protein was precipitated at 4°C for 20 min. The supernatant was collected by centrifugation, filtered through a microporous filter, and stored at -80°C.

[0154] The samples were tested using HPLC.

[0155] The results showed that compound Ⅰ-1 was incubated in rat plasma at 37°C for 24 hours, and HPLC detection showed no significant decrease in peak area, indicating good stability.

[0156] Example 34: Application in the preparation of drugs or pharmaceutical compositions for treating and preventing inflammatory bowel disease, sepsis and other related diseases.

[0157] Prepare the following reagents by conventional methods

[0158] tablet

[0159]

[0160]

[0161] The above formula is prepared into tablets using conventional methods.

[0162] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention herein is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also encompasses other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the inventive concept. For example, a technical solution formed by replacing the above-mentioned features with, but not limited to, technical features having similar functions disclosed in this application.

Claims

1. A ginsenoside derivative as represented by formula I, II, III, IV, V or VI, or a pharmaceutically acceptable salt of a ginsenoside derivative as represented by formula I, II, III, IV, V or VI In formula I, II, III, IV, V or VI, R is 4-nitrophenyl, 4-nitrilephenyl or 3-pyridyl.

2. The ginsenoside derivative according to claim 1, characterized in that The ginsenoside derivatives are 20(S)-12β, 20-dihydroxy-2-(4-nitrobenzylidene)-dammarane-24-ene-3-one, 20(S)-12β, 20-dihydroxy-2-(4-cyanobenzylidene)-dammarane-24-ene-3-one, 20(S)-12β, 20-dihydroxy-2-(3-pyridylmethylene)-dammarane-24-ene-3-one, (20S, 24R)-epoxy-12β, 25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one, (20S, 24R)-epoxy-12β, 25-dihydroxy-2-(4-cyanobenzylidene)-dammarane-3-one, (20S, 24R)-epoxy-12β, 25-dihydroxy-2-(4-cyanobenzylidene)-dammarane-3-one, and (20S, 24R)-epoxy-1 2β,25-dihydroxy-2-(3-pyridylmethylene)-dammarane-3-one, (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one, (20S,24S)-epoxy-12β,25-dihydroxy-2-(4-nitrobenzylidene)-dammarane-3-one, 20(S)-dammarane-2-(4-nitrobenzylidene)-24-ene-3β-12β,20-triol, 20(S)-dammarane-2-(4-nitrobenzylidene)-24-ene-3β-12β,20-triol or (20S,24R)-epoxydammarane-2-(4-nitrobenzylidene)-3β,12β,25-triol.

3. The method for preparing the ginsenoside derivatives of formula I, II, III, IV, V or VI according to claim 1, characterized in that The synthetic route of the method is shown below:

4. The method according to claim 3, wherein The method comprises the following steps: (1) subjecting protopanaxadiol, the 24R-pyxinol ginsenoside derivative shown in Formula 3, or the 24S-pyxinol ginsenoside derivative shown in Formula 4 to a selective oxidation reaction under the action of an oxidant to obtain 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one shown in Formula 1, the intermediate product shown in Formula 5, or the intermediate product shown in Formula 7, respectively; (2) 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one represented by Formula 1, the intermediate product represented by Formula 5, or the intermediate product represented by Formula 7 is subjected to a condensation reaction with an aldehyde R-CHO in an alcoholic solution of potassium hydroxide to prepare a ginsenoside derivative represented by Formula I, II, or III, respectively; (3) The ginsenoside derivatives represented by formula I, II or III are subjected to a reduction reaction in an alcohol solution of NaBH4 to obtain the ginsenoside derivatives represented by formula IV, V or VI, respectively.

5. The method according to claim 4, wherein In the step (1), the oxidant is Dess-Martin reagent or PCC; the molar ratio of protopanaxadiol, the 24R-pyxinol ginsenoside derivative shown in formula 3 or the 24S-pyxinol ginsenoside derivative shown in formula 4 to the Dess-Martin reagent is 1:1 to 1.

2.

6. The method according to claim 4, wherein In the step (2), the molar ratio of 20(S)-12β,20-dihydroxy-dammarane-24-ene-3-one represented by formula 1, the intermediate product represented by formula 5 or the intermediate product represented by formula 7, and the aldehyde R-CHO is 1:0.1-10.

7. The method according to claim 4, wherein In the step (3), the molar ratio of the ginsenoside derivative represented by formula I, II or III and NaBH4 is 1:1-10.

8. Use of the ginsenoside derivatives represented by formula I, II, III, IV, V or VI and pharmaceutically acceptable salts thereof as claimed in claim 1 in the preparation of anti-inflammatory drugs or anti-inflammatory pharmaceutical compositions.

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